Biomedical subjects

E W Rubel

Publications and source records attributed to E W Rubel.

At least 91 records · Page 5Linked to original sources

PubMed1994

Diffusible factors regulate hair cell regeneration in the avian inner ear.

Damage to the avian inner ear results in up-regulation of mitotic activity resulting in regeneration of hair cells. The objective of this investigation was to determine whether the damaged inner ear epithelium releases a soluble mitogen that is responsible for the up-regulation of proliferation. The sensory epithelium from normal and drug-damaged avian inner ears was cultured alone or in the presence of other cultures. As previously shown in vivo and in vitro, damaged organs displayed increased supporting cell proliferation compared with undamaged organs, leading to eventual morphologic and functional recovery. When damaged organs were cocultured with an undamaged organ, proliferation was increased in the undamaged tissue. When undamaged organs were cultured together, proliferation was decreased. These results indicate that a soluble factor released from the damaged inner ear epithelium stimulates proliferation and suggest the release of a factor from normal tissue that suppressed mitotic activity. Thus, reparative hair cell regeneration in the inner ear appears to be regulated by a balance between proliferative and antiproliferative paracrine factors.

Animals↗

PubMed1994

A characteristics protein highly expressed in guinea pig inner ear, defined by monoclonal antibody WH-1.

A new monoclonal antibody (termed WH-1; isotype IgG2b) was established using a homogenate of dissected guinea pig cochleas (N = 60) as immunogen. Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) identified the WH-1 antigen as a protein or glycoprotein with M(r) approximately 40 kDa. Immunoperoxidase treatment of histologic cryosections of guinea pig cochlea, followed by light microscopic examination, revealed strong positive staining at three sites: (i) parts of Hensen's stripe, marginal band, covering net, and Kimura's membrane (within the tectorial membrane [TM]); (ii) Deiters' cells, pillar cells, Hensen's cells, and stereocilia of outer hair cells (within the organ of Corti); (iii) interdental cells, inner and outer sulcus cells, Reissner's membrane, and surface membrane of stria vascularis epithelium. Similar staining patterns were observed for cryosections of rat and mouse cochleas. Only a trace quantity of cross-reacting protein was detectable in brainstem. The protein was not detectable in tongue extract by Western blotting. However, sections of brainstem and tongue did show positive immunohistological staining with WH-1. Localization of WH-1 antigen was further examined by electron microscopy. WH-1 positivity on outer hair cell stereocilia, certain sites on the TM, interdental cell surface, Reissner's membrane epithelia, and inner and outer sulcus cells was confirmed. WH-1 antigen was not detected on inner hair cell stereocilia by light or electron microscopy. The localization of WH-1 antigen on outer hair cell stereocilia and TM suggests that it may play some role in adhesion between these structures.

Animals↗

PubMed1994

Hair cell regeneration in the inner ear.

Hearing and balance disorders caused by the loss of inner ear hair cells is a common problem encountered in otolaryngology-head and neck surgery. The postembryonic production of hair cells in cold-blooded vertebrates has been known for several decades, and recent studies in the avian inner ear after ototoxic drug and noise damage have demonstrated a remarkable capacity for both anatomic and functional recovery. The regeneration of sensory hair cells has been shown to be integral to this repair process. Current work is focusing on the cellular progenitor source of new hair cells and the trigger mechanism responsible for inducing hair cell regeneration. Preliminary studies suggest that reparative proliferation may also occur in the mammalian inner ear. Work in this field is moving at a rapid pace. The results thus far have yielded optimism that direct stimulation of hair cell production or transplantation of living hair cells may eventually become treatment modalities for the damaged human inner ear. These proposals would have been considered unrealistic less than 10 years ago, but they now have caught the full attention of both clinician and researcher.

Animals↗

PubMed1994

Identification of hair cell progenitors and intermitotic migration of their nuclei in the normal and regenerating avian inner ear.

Postembryonic production of sensory hair cells occurs in both normal and aminoglycoside-damaged avian inner ears. The cellular source and mechanism that results in new differentiated hair cells were investigated in the avian vestibular epithelia using three distinct cell-cycle-specific labeling methods to identify proliferating sensory epithelial cells. First, immunocytochemical detection of the proliferating cell nuclear antigen, an auxiliary protein of DNA polymerase, allowed labeling of cells in late G1, S, and early G2 phases of the cell cycle. Second, a pulse-fix tritiated thymidine autoradiographic protocol was used to identify cells in S phase of the cell cycle. Finally, Hoechst 33342, a fluorescent DNA stain, was used to identify epithelial cells in mitosis. The distribution of cells active in the cell cycle within the normal and ototoxin-damaged vestibular epithelium suggests that supporting cells within the sensory epithelia are the cellular precursors to the regenerated hair cells. Differences between the proliferation marker densities in control and damaged end organs indicate that the upregulation of mitotic activity observed after streptomycin treatment is due primarily to an increase in the number of dividing progenitor cells. The differences between the extent of ototoxic damage and the level of reparative proliferative response suggest a generalized stimulus, such as a soluble chemical factor, plays a role in initiating regeneration. Finally, after DNA replication is initiated, progenitor cell nuclei migrate from their original location close to the basement membrane to the lumenal surface, where cell division occurs. This pattern of intermitotic nuclear migration is analogous to that observed in the developing inner ear and neural epithelium.

Aminoglycosides↗

PubMed1994

The astrocytic response to afferent activity blockade in chick nucleus magnocellularis is independent of synaptic activation, age, and neuronal survival.

Astrocytes in nucleus magnocellularis (NM) of the chick respond to afferent activity blockade with increased immunoreactivity for glial fibrillary acidic protein (GFAP). NM neurons respond to the same manipulations with reduced protein synthesis, ribosomal dissociation, and subsequent death of a subset of these neurons. In the present study, we sought to evaluate the relationship between these neuronal and glial responses and to determine if similar activity-dependent mechanisms mediate them. We first examined the anatomical relationship between NM neurons and astrocytic processes by electron microscopy and GFAP immunostaining. Both methods showed that NM neurons deprived of activity for 6 hr were apposed by more glial processes than active NM neurons. However, we found no preferential positioning of GFAP-immunoreactive processes near neurons of the dying or surviving populations, and there were no differences in glial process apposition to dying versus surviving neurons at the EM level. To determine whether the astrocytic response is similar to the neuronal response in age dependence, GFAP immunoreactivity was analyzed in adult chickens following unilateral afferent activity blockade. Unlike the neuronal response to activity blockade, the astrocytic response is equally strong in adult animals. These results imply an independence of the neuronal and astrocytic responses to activity blockade, raising the possibility that these two cell types may be responding to different activity-related signals. This possibility was tested using an in vitro slice preparation. Unilateral stimulation of NM was provided in three ways: orthodromically, antidromically, and orthodromically in a low-calcium medium. The regulation of astrocytic GFAP immunoreactivity by these manipulations of activity was then analyzed. The results of these experiments show that, unlike neuronal protein synthesis, astrocytic GFAP immunoreactivity can be suppressed by either presynaptic or postsynaptic neuronal activity. Therefore, the astrocytes and neurons are regulated by different activity-dependent signals and, by the present measures, their responses to activity blockade appear independent of one another.

Aging↗

PubMed1994

Membrane properties underlying the firing of neurons in the avian cochlear nucleus.

Neurons of the avian nucleus magnocellularis (NM) relay auditory information from the VIIIth nerve to other parts of the auditory system. To examine the cellular properties that permit NM neurons to transmit reliably the temporal characteristics of the acoustic stimulus, we performed whole-cell recordings in neurons of the chick NM using an in vitro thin slice preparation. NM neurons exhibited strong outward rectification near resting potential; the voltage responses to depolarizing current steps were substantially smaller than to equivalent hyperpolarizing steps. Suprathreshold current steps evoked only a single action potential at the start of the step. In contrast, stimulation with trains of brief current pulses evoked repetitive firing that was phase-locked to the stimulus cycle. The number of action potentials evoked by the pulses during the train decreased with increasing stimulus rate. Voltage-clamp experiments revealed a rapidly activating, slowly inactivating, outward current with a threshold near -65 mV. During depolarizing voltage steps, the outward current rose sigmoidally to a peak and then decayed slowly, reaching steady state within 5 sec. Application of 200 microM 4-aminopyridine (4-AP) reduced the peak of the outward current by 84%, leaving a small, persistent component. Under current clamp, application of 200 microM 4-AP reduced the outward rectification and increased the amplitude and duration of the action potentials. Moreover, NM neurons could no longer sustain firing during high rates of stimulation with the current pulses: increased temporal summation of the potentials caused sufficient depolarization to inactivate the sodium conductance underlying the action potential. These results suggest that the outward current is necessary for NM neurons to transmit well-timed events reliably for the duration of an acoustic stimulus.

4-Aminopyridine↗

PubMed1994

Cell division in the gerbil cochlea after acoustic trauma.

The recent discovery of hair cell regeneration in the avian inner ear raises the possibility that hair cell regeneration might occur in the mammalian cochlea as well. The authors used 3H-thymidine labeling to detect mitotic activity in the cochleas of normal 3-week old gerbils exposed to acoustic trauma. Following an acoustic insult that caused progressively more severe damage in an apical to basal progression, 3H-thymidine was injected for 5 days. Control animals were not exposed to the acoustic insult. The gerbils' cochleas were sectioned and processed for autoradiography. In the control cochleas, there were extremely rare labeled cells in the stria, the spiral ligament, and the glial cells around the acoustic nerve fibers. In the damaged cochleas, no evidence of hair cell regeneration or of any cell division within the normal sensory epithelial structures was seen. Three labeled cells were seen in intercellular spaces within the sensory epithelium; they appeared to be macrophages. Frequent cell division was seen in numerous other regions of the damaged cochleas and among glial cells adjacent to the acoustic nerve fibers. It is concluded that there is no evidence for hair cell regeneration following acoustic trauma in the gerbil, but acoustic trauma does induce cell division in numerous other areas of the cochlea.

Animals↗

PubMed1994

A biphasic change in ribosomal conformation during transneuronal degeneration is altered by inhibition of mitochondrial, but not cytoplasmic protein synthesis.

Following loss of eighth nerve input, 20-40% of neurons in the neonatal chick cochlear nucleus, nucleus magnocellularis (NM), undergo cell death. Intracellular changes that precede the death of NM neurons include increased oxidative metabolism and mitochondrial volume, decreased cytoplasmic protein synthesis, and destruction of ribosomes. Six hours following afferent deprivation, dying NM neurons demonstrate complete loss of ribosomes and cessation of protein synthesis, suggesting that the rapid destruction of ribosomes leads to neuronal death. Increased NM neuron death occurs when mitochondrial upregulation is prevented by chloramphenicol, a mitochondrial protein synthesis inhibitor. This finding suggests that increased oxidative capacity is required for neuronal survival following loss of afferent input. To study changes in the ribosomes of afferent-deprived NM neurons, we obtained a monoclonal antibody to ribosomal RNA. This monoclonal antibody, Y10B, labels ribosomes of all NM neurons receiving normal synaptic activity. Following removal of afferent input, NM neurons demonstrate a biphasic change in their pattern of Y10B label. During the initial phase, there is a uniform decrease in the density of Y10B label. In the second phase, some NM neurons recover the capacity to bind the Y10B antibody while others remain unlabeled. During this second phase, NM neurons putatively destined to die, based on their failure to synthesize protein, are unlabeled by the Y10B antibody. New gene expression is not necessary to initiate the change in ribosomal immunoreactivity that leads deafferented NM neurons toward cell death. Blocking cytoplasmic protein synthesis with cycloheximide had no effect on the biphasic change in Y10B labeling of afferent-deprived NM neurons. Treating chicks with chloramphenicol, however, prevented the recovery of Y10B immunoreactivity in NM neurons during the second phase of the response to afferent deprivation.

Afferent Pathways↗

PubMed1993

Morphological correlates of functional recovery in the chicken inner ear after gentamycin treatment.

Newly hatched chickens were allowed to survive 6, 10, 15, and 20 weeks after 10 days of gentamycin sulfate treatment. Ultrastructural studies of hair cells and nerve terminals in the auditory receptor organ, the basilar papilla, were carried out with transmission and scanning electron microscopes. Attention was paid to absolute sensory cell (hair cell) numbers, stereocilia maturity and orientation, and reinnervation within a band 100 micron wide centered 1,100 microns from the basal end of the avian cochlea. Sensory cell numbers were equivalent to those of untreated control animals within the study area in the earliest survival group. Both immature and mature appearing hair cells were identified throughout the recovery period. However, the ratio of mature to immature hair cells gradually increased to exceed 95% at 20 weeks. Stereocilia bundle reorientation also occurred throughout the study period. Orientation was often abnormal at 6 weeks, but by 20 weeks more than 95% of the regenerated hair cells were aligned within normal limits established in the control ears. Hair cell differentiation occurring at 10-15 weeks was associated with degeneration of the afferent nerve receptor complexes commonly observed in 6 week survivors. These complexes were replaced by one or two small bouton shaped efferent terminals per cell. At 20 weeks, two or three chalice shaped vesiculated terminals were observed per cell in both the gentamycin treated and control ears. On the basis of these observations normal physiological activity would be predicted at 20 weeks following gentamycin treatment, at which time sensory cell repopulation, maturation, reorientation, and innervation approximates the normal anatomical condition.

Animals↗

PubMed1993

Hair cell regeneration after streptomycin toxicity in the avian vestibular epithelium.

Recent reports documented the ability of the posthatch avian vestibular epithelia to produce hair cells continually at a low rate. This project was designed to investigate whether, in addition, the chicken vestibular system is capable of regenerating its sensory epithelium in response to a lesion. Aminoglycoside injections were given to young birds in order to damage the vestibular epithelium. Tritiated thymidine injections were used to label cells produced in response to the lesion. Treatment and age-matched control animals were killed at 1 day, 20 days, or 60 days after aminoglycoside injections, and vestibular organs were processed for autoradiography. Our results show that the chicken vestibular sensory epithelium is capable of regenerating hair cells after severe damage. Moreover, the epithelium is capable of complete anatomical recovery. Finally, drug damage increases the pace at which hair cells are replaced, compared to the rate of hair cell turnover in untreated tissue.

Animals↗

PubMed1993

Hair cell regeneration in the European starling (Sturnus vulgaris): recovery of pure-tone detection thresholds.

Behavioral detection thresholds were obtained from four starlings before, during, and after 11 days of subcutaneous injections of kanamycin. Birds were operantly conditioned to respond to pure-tones ranging in frequency from 0.25 kHz to 7 kHz using the method of constant stimuli and were tested daily for 141 days after the first injection of aminoglycoside. All four birds sustained hearing losses greater than 60 dB at frequencies from 4 kHz to 7 kHz by the end of the 11 day injection schedule. Two birds had a slight shift in threshold at 3 kHz. No change in threshold occurred for any of the birds at lower frequencies. Recovery of detection thresholds began soon after the injections ceased and continued for approximately 50 days. In all four birds there was some degree of permanent hearing loss: 5 dB to 15 dB at frequencies between 4 kHz and 6 kHz, and approximately 25 dB at 7 kHz. Scanning electron microscopy (SEM) was performed at 0 and 5 days post-injection in a separate group of starlings given the same injection schedule. Hair cell loss and damage was observed across the basal 34% to 36% of the basilar papilla. SEM in two behaviorally tested birds sacrificed 142 days after the first injection showed that there was regeneration of hair cells to populate the previously damaged region, but that disorientation of stereocilia bundles in the basal third of the basilar papilla was common. The other two behaviorally tested birds were treated with kanamycin again for 16 days beginning at 142 days after the first injection. Thresholds shifted again, but less than during the first dosing period. SEM of these birds' basilar papillae showed less hair cell loss than observed in the birds given only a single, 11 day dosing of kanamycin. This result suggests that birds may be less susceptible to the ototoxic effects of kanamycin in repeated treatments. In all four birds, the degree and position of damage observed with SEM corresponded with the extent and frequency of hearing loss.

Acoustic Stimulation↗

PubMed1993

Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.

The sensory epithelium of the avian inner ear retains into adulthood progenitor cells for inner-ear hair cells and other cell types in the epithelium. Hair cells are produced normally on an ongoing basis in the vestibular sensory epithelium, and hair-cell production is increased after insult in both auditory and vestibular sensory epithelia. The details of postnatal hair-cell production are not understood. In particular, molecular factors involved in the initiation and regulation of hair-cell genesis and differentiation are not known. Studies of this phenomena have been hampered by the lack of cell culture models. An organ culture system was developed which encourages generation and differentiation of hair cells in mature inner-ear sensory epithelia. Continuous labeling with tritiated thymidine showed genesis of both supporting cells and hair cells in normal vestibular epithelia grown in culture, and an increase in hair-cell and supporting-cell proliferation in damaged sensory epithelia grown in culture as compared to undamaged controls. This demonstrates, in vitro, both the division and differentiation of hair-cell progenitor cells in normal vestibular epithelia, and the maintenance of the hair-cell regeneration process in damaged inner-ear epithelia. This culture system should be useful for studies of hair-cell genesis and differentiation as well as studies of hair-cell and supporting-cell functioning in general.

Animals↗

PubMed1993

Postnatal production of supporting cells in the chick cochlea.

The auditory receptor organ in birds, the basilar papilla, is mitotically active after acoustic overstimulation or pharmacological insult and is capable of self-repair. The damaged epithelium is repopulated with new hair cells and supporting cells. The cell production that underlies this regenerative self-repair is believed to be a response evoked by damage in populations of cells that normally become mitotically quiescent even before hatching. In contrast, regeneration in the vertebrate nervous system is often correlated with continued or recent neurogenesis in the tissue concerned. The hypothesis that there may be ongoing postnatal production of cells in the normal avian basilar papilla was investigated. Autoradiographic analysis of tritiated-thymidine-injected animals was used to look for the existence of newly formed cells in the basilar papilla of normal posthatch chickens. Several types of supporting cells, namely, organ supporting cells, border cells and hyaline cells, are produced postnatally in the normal chicken. Typically, they are added interstitially to the apical (distal) half of the basilar papilla.

Acoustic Stimulation↗

PubMed1993

Altered malate dehydrogenase activity in nucleus magnocellularis of the chicken following cochlea removal.

The metabolism of second order auditory neurons in nucleus magnocellularis (NM) in the chick brainstem can be profoundly altered when excitatory input from the cochlea is removed. Within hours of cochlea removal, NM neurons show an increase in histochemical staining for the Kreb's cycle enzyme succinate dehydrogenase (SDH), followed in several days by decreases in SDH staining. We examined the activity of another Kreb's cycle enzyme, malate dehydrogenase (MDH) using a histochemical stain and a sensitive quantitative biochemical assay for comparison. We found changes in MDH staining similar in time course and magnitude to those of SDH; within 4 h of cochlea removal, MDH activity increases in ipsilateral NM neurons. By 9 days after cochlea removal MDH activity decreases, although not to the same degree as seen with SDH. Biochemical measurements of MDH activity also showed an early increase in activity in ipsilateral NM, followed at 9 days survival by a decrease in activity. Biochemical measurements of the activity of other enzymes in NM may be useful in further defining the metabolic consequences of deafferentation.

Animals↗

PubMed1993

Vulnerability and adaptation of distortion product otoacoustic emissions to endocochlear potential variation.

The endocochlear potential (EP) was reversibly decreased in adult gerbils by the intraperitoneal injection of furosemide, while cochlear functioning was monitored by measurement of distortion production otoacoustic emissions (DPE) at a range of stimulus intensities. Stimulus frequencies for DPEs were f1 = 6.8 and f2 = 8 kHz (f2/f1 = 1.18). Emissions monitored in the ear canal and scala media were 2f1-f2, 3f1-2f2, 2f2-f1, and f2-f1. Typically, the EP decreased smoothly, reached a minimum one-half hour after injection, then recovered slowly over several hours. Emissions at 2f1-f2 and 3f1-2f2 at low stimulus levels were particularly vulnerable to the change in EP. These vulnerable emissions showed characteristic trajectories in which the amplitudes changed little with the initial EP decrease, then dropped sharply as the EP continued to decrease. However, the amplitudes then began to recover even before the EP reached minimum, and recovered completely while the EP remained subnormal. The trajectories of the other odd order emissions were similar, but lacked the abrupt decrease. The variation of the even order (f2-f1) component was completely different, but appeared related to the odd order trajectories in a complex fashion. During the initial decrease for the vulnerable components, the decrease in emission amplitude (in dB) was found to be proportional to the square of the change in EP (in mV). The recovery with a subnormal EP was interpreted as an adaptive effect with a time constant of about 15 min.

Acoustic Stimulation↗

PubMed1993

Current concepts in hair cell regeneration.

Hair cell regeneration following various mechanical or chemical insults to the inner ear has become the subject of intense scientific research. This article details current concepts relating to this topic, including descriptions of the hair cell precursor population, the ultrastructural properties of the newly regenerated hair cells, and regeneration of hair cells on the vestibular epithelium. Numerous photomicrographs supplement the text.

Animals↗

PubMed1992

Rapid changes in protein synthesis and cell size in the cochlear nucleus following eighth nerve activity blockade or cochlea ablation.

Destruction of the cochlea causes secondary changes in the central auditory pathway through transynaptic regulation. These changes appear to be mediated by an activity-dependent process and can be detected in the avian auditory system as early as 30 minutes after deafferentation. We compared the early changes in cochlear nucleus neurons following deafferentation by cochlea ablation with those seen following activity deprivation by perilymphatic tetrodotoxin (TTX) exposure. Protein synthesis and size of large spherical cells in the anteroventral cochlear nucleus (AVCN) of 14-day-old gerbils were measured during the first 48 hours after the manipulations. Both cochlea ablation and TTX produced a reliable decrease in protein synthesis by AVCN neurons (30-40%) by 1 hour. The magnitude of change in tritiated leucine incorporation was similar at all survival times, in both experimental groups. In contrast to the rapid changes in protein synthesis, the decrease in cell size was first evident 18 hours after TTX exposure and 48 hours after cochlea ablation. There was no significant change in protein synthesis or cell size in control groups at any of the survival times. These findings are consistent with changes in the avian auditory system in response to deafferentation and TTX exposure. Cochlea ablation and TTX exposure induced similar transneuronal changes, supporting the hypotheses that activity of auditory afferents in young mammals plays a regulatory role in the metabolism and morphology of their target neurons in the central auditory pathway, and that early changes following destruction of the peripheral receptor are due to reduction of activity-dependent interactions of presynaptic and postsynaptic cells.

Analysis of Variance↗

PubMed1992

Rapid growth of astrocytic processes in N. magnocellularis following cochlea removal.

Removal of the cochlea or pharmacological blockade of eighth nerve activity in young postnatal chickens results in rapid transneuronal cell death and atrophy in neurons of n. magnocellularis. The present experiments were designed to examine the influence of afferent input on astrocyte structure in n. magnocellularis. Young chickens were subjected to unilateral cochlea removal. At times ranging from 5 minutes to 72 hours later, the brainstems were histologically processed with a polyclonal antibody against glial fibrillary acidic protein (GFAP). A second group of chick brainstems was impregnated by a Golgi method 6 hours after unilateral cochlea removal and impregnated three-dimensional reconstructions were made of glial cells in n. magnocellularis (NM). Analyses of GFAP positive processes in NM revealed an observable increase in the number of astrocytic processes at the borders of the nucleus within 30 minutes of cochlea removal and a twofold increase in GFAP + glial processes by 6 hours. A secondary increase in the number and density of GFAP + processes occurred between 24 and 72 hours following cochlea removal, during the period of axonal degeneration, and transneuronal cell atrophy and death. Analyses of astrocytes impregnated by the Golgi method revealed that individual glial cells had increased their total process length and the number of processes by approximately twofold by 6 hours after cochlea removal. These results suggest that the structure of astrocytes is rapidly and dramatically influenced by the level of excitatory activity in a neuronal system. Furthermore, the similarity of results obtained with GFAP immunohistochemistry and three-dimensional reconstruction of astrocytes provides evidence that the short-term changes observed following cochlea removal represent the actual growth of glial processes. We speculate that modulations in glial processes as a function of afferent activity may act to influence synaptic efficacy.

Afferent Pathways↗